The first genetic defect responsible for impaired tolerance induction in SLE was recently identified in MRL mice. The mutation in the Fas gene leads to accumulation of double negative (DN) T cells and impaired tolerance. The mechanisms by which these abnormalities occur are not known. In this application we plan to investigate these questions as follows: 1. To determine whether B220+ DN cells originate from cells that have previously expressed CD4 and/or CD8 or from an alternative developmental pathway, we will examine: precursor-product relationships of lpr thymocytes ; the ontogeny of B220+ DN T cells; the transcriptional status of CD4 and CD8 molecules in B220+ DN T cells and the role of each major T cell subset in the induction of lupus 2. To determine the time, site, and mechanism of action of Fas we will analyze the exact stage at which Fas is expressed on developing thymocytes using a monospecific rabbit anti-Fas antibody. The functional consequences of the Fas defect will be determined by comparing apoptosis of lpr and + + lymphocytes in response to antigen receptor stimulation. Negative selection and maintenance of tolerance in lpr TcR Tg mice bearing TcR specific for antigen will be compared to Tg mice without the Fas defect. The role of the lpr mutation in B cells and T cells at different stages of differentiation will be investigated. 3. To determine whether Fas or other functionally related proteins play a role in the pathogenesis of SLE in non-lpr mice, the expression of Fas and Bc1-2 in lymphoid tissues and lymphocyte populations will be quantified in lupus strains in the pre- and autoimmune phases of the disease. 4. We will attempt to clone the Fas ligand to determine its potential use as a therapeutic reagent. These studies will provide essential insight into the role of the Fas receptor in regulating self/nonself discrimination. This information will provide new ideas for intervention in SLE.